Almost all animals from sea urchins to fruit flies, to fish and frogs, to mice and men undergo a process called gastrulation in their early embryonic life. During gastrulation, pluripotent cells involute to the centre of the embryo. The cells allocated in this manner are destined to form layers of more specialized embryonic cells, the mesoderm and the endoderm. The mesoderm will form blood and the musculoskeletal system. The endoderm will form the digestive tract and associated internal organs.
Shortly following gastrulation, the embryo undergoes neurulation. In vertebrates, the hallmark of neurulation is the establishment of the neural plate from pluripotent cells. The neural plate bends ventrally at its midline causing the edges to come together and form a tube. All of the cells of the central and peripheral nervous system are derived from the neural plate and neural tube. The cells of the neural plate are the first cells to become committed to the neural lineage.
The ability to study these early neural cells in more detail would provide valuable information relating to numerous neurodevelopmental and neurodegenerative disorders. At present, over ten million people, worldwide are afflicted by neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis, and Huntington's Disease. Thus, there is considerable interest in the development of stem cell models and therapies to remedy the effects of neurodegenerative disease.
Neural cell types can be generated from pluripotent stem cells; however, the differentiation of embryonic stem cells requires multiple steps and does not often efficiently generate the specific neurons lost in neurodegenerative disease. An attractive alternative would be stem cell lines already restricted to the neural lineage that could be rapidly differentiated to neurons of clinical interest. In addition, cultured neural stem cell lines would provide a means to study human neurodevelopment.
Derivation of Neural Stem Cell Lines
Evidence that neural stem cells could be maintained in vitro was provided by Reynolds Tetzlaff and Weiss 1992; culture of 14 day old striata with the growth factor EGF was reported to lead to the propagation of stem-cell comprising floating cell clusters (“neurospheres”). However, neurospheres consist predominately of committed neural progenitors and differentiated cells, with the maintained stem cells not being directly identifiable or purified. Moreover, the stem cells maintained in neurospheres have an uncertain relationship to CNS precursor cells in vivo.
Later studies showed that cells within the neurospheres were responsive to bFGF (Vescovi et al 1993), with the application of bFGF leading to the proliferation of two progenitor cell types. Further experiments demonstrated that neural precursor cells could be propagated from adult mouse striatum by culture with bFGF (Gritti et al 1996), from the lumbar/sacral segment of the spinal cord with EGF+bFGF (Weiss et al 1996), and in adherent cultures when cultured with the growth factor FGF2 (Johe et al 1996).
Rathjen et al. 2002 (and also in U.S. patent application Ser. No. 10/090,849; publication number US2002/0151054 A1) report the derivation of a neuroectodermal lineage which is described as “multipotential” and having the capacity to differentiate into a number of neuronal cell types, including neuronal cells, glial cells and neural crest cells. However, the cells described in Rathjen are shown to express the neurogenic bHLH factor mash1, and the marker of BMP-2 activity, pax3. Pax3, in particular, is an indicator of dorsoventral patterning, indicating that the cells in Rathjen are partially differentiated.
Conti et al 2005 report the derivation and maintenance of a neural stem cell (NS cell) monoculture through culturing cells in N2 media supplemented with the growth factors FGF2 and EGF. Conti et al report that the addition of both FGF 2 and EGF is critical for the continued propagation of the NS cells which they had derived.
The NS cells' characteristics indicate they are closely related to the radial glia lineage, with uniform and stable expression of neurogenic basic Helix-loop-Helix (bHLH) factors such as olig2 and mash1, and limited ability to differentiate into neuronal cell types.
The Role of FGF2 and FGF4
In addition to the above described role in promoting the proliferation of neural progenitor cells, FGF2 has also been reported to promote the differentiation of embryonic stem cell into neural fates. For example, Forsberg et al 2012 report that the addition of FGF2 and heparin to NDST 1/2 negative murine ES cells restored the ability of the ES cells to differentiate into neural cell types. The ability to restore neuronal differentiation was also seen on addition of FGF4 and heparin.
FGF2 and FGF4 have also been reported to induce proliferation of cells in an in vitro neurosphere assay (Kosaka et al 2006). In this assay, primary germinal zone cells from the ganglionic eminence of an E14 mouse embryo were cultured in the presence of either FGF2 or FGF4. It was found that both FGF2 and FGF4 led to an increase in the number of neurospheres which were formed, along with an increase in cell viability.
In the same paper, Kosaka et al also report that FGF4 induces differentiation of EGF-responsive stem-cell progeny in a manner comparable to that of FGF2, leading them to propose FGF4 as a key inducer of neuronal differentiation. This latter proposition is consistent with the results reported by Chen et al 2010, where both FGF4 and FGF2 are shown to lead to significantly elevated neural induction in ‘46C’ mouse ES cells (GFP-Sox1 knock-in).
As well as its reported function in promoting the differentiation of neural cell types from ES cells, FGF4 has been reported to promote the maintenance of some (non-neural) stem cell types.
Tanaka et al 1998 reported the isolation of permanent trophobalst stem (TS) cell lines by culturing mouse blastocysts or early post-implantation trophoblasts in the presence of FGF4. This result was subsequently confirmed by Abell et al 2009, who further characterized the mechanism by which FGF4 maintains TS cells as employing the MEKK4 kinase as a signalling hub.
FGF4 has also been reported to support the undifferentiated growth of human ES cells (Mayshar et al 2008). Targeted knockdown of FGF4 expression in these cells was observed to lead to increased differentiation of the hES cells.
However, as indicated above, the differentiation of embryonic stem cells requires multiple steps and does not often efficiently generate the specific neurons that are sought. Of the neural stem cell lines currently available, none is of a sufficiently early developmental stage to allow differentiation into every neural cell type. For example, NS cells arederived from embryonic day 12 (E12) onwards, whereas dopaminergic neurons (of interest in Parkinsons disease, for example) are already differentiated by stage E11.5 (nb. Staging relates to mouse embryos). In addition, a neural stem cell line from a very early stage would allow the modelling of early-stage neural defects, such as neural tube defects (NTDs). Finally, the availability of an early neural stem cell line would give researchers much more control over the process of neural development and the ability to study and direct differentiation.
Thus, there is a need for the development of a neural stem cell line from an early stage of neural development which is capable of differentiation into a broad range of neuronal subtypes and glia.